PTEN encodes a phosphatidylinositol-3-phosphate phosphatase and is inactivated in over 30% of human cancer. The major goal of this project is to investigate the mechanism of action of PTEN at the cell junction. Loss of cell junctional integrity is a primary feature of metastatic tumors. Understanding the signaling events controlled by PTEN will lead to the identification of molecular targets for therapeutic interventions. This project is built on the important findings that PTEN is a phosphoprotein and certain phosphorylated forms of PTEN show a greater binding capacity to a scaffolding protein, MAGI-2, at the cell junction. In Specific Aim 1, the biological and biochemical properties of PTEN phosphoisoforms will be examined. A panel of PTEN phosphorylation site mutants will first be generated by site-directed mutagenesis. Their relative phosphatase activity will be assayed using water-soluble fluorescent phosphoinositides as substrates. Also, their subcellular localization will be determined by indirect immunofluorescence analysis. In Specific Aim 2, the tumor suppressing activity of MAGI-2 will be examined. The ability of MAGI-2 to alter survival, cell-cell adhesion, growth, cell motility, and tumorigenicity will be investigated using human tumor cells which lack the expression of MAGI-2. Whether the interaction between PTEN and MAGI-2 is necessary for tumor suppression will be tested using small interfering (si) RNA to down-regulate PTEN expression. In Specific Aim 3, the signaling molecules responsible for the biological functions of MAGI-2 will be delineated. The role of 13-catenin in mediating MAGI-2 actions will be tested by measuring its subcellular distribution through indirect immunofluorescence analysis. Also, 13-catenindependent transcriptional responses will be assessed by luciferase-based reporter assays using a TCF/Lef promoter element. In summary, this application focuses on an exciting and under-explored area of research into the function of PTEN at the cell junction. The proposed studies will provide mechanistic models that can be used to explain such critical physiological phenomenon as contact inhibition. In addition, novel signaling pathways will be delineated and will provide targets for tumor diagnosis and therapeutic interventions.